FIG. 5.

p204 can overcome the inhibition of the sequence-specific binding to DNA of MyoD, E47, and the MyoD-E47 heterodimer by the Id proteins. (A) Id2, Id1, and Id3 inhibit the sequence-specific binding of MyoD to the MEF-1 oligodeoxynucleotide in vitro, and p204 overcomes the inhibition, as determined by EMSA. The proteins indicated, i.e., GST (0.5 μg); GST-MyoD (0.5 μg); Id2, Id1, or Id3 translated in a reticulocyte lysate (IVT-Id2, IVT-Id1, and IVT-Id3, respectively) (0.5 μg); and GST-204 (1 μg), were mixed in the reaction buffer (20 μl). For competition experiments, a 50-fold excess of wild-type or mutant MEF-1 oligodeoxynucleotide was added. For supershift assays, anti-MyoD IgG (0.5 μg) was included. After 15 min of incubation the ³²P-MEF-1 probe was added, and the reaction mixture was incubated for a further 20 min and analyzed by gel electrophoresis. The positions of the supershifted MyoD-DNA complex (arrow 1), the MyoD-DNA complex (arrow 2), and the free DNA probe (arrow 3) are indicated. (B) Id2 inhibits the sequence-specific binding of E47 homodimers and MyoD-E47 heterodimers to the MEF-1 oligodeoxynucleotide in vitro, and p204 overcomes the inhibition. The proteins indicated, i.e., GST (0.5 μg), GST-MyoD (0.5 μg), and Id2 translated in a reticulocyte lysate (0.5 μg), were mixed in the reaction buffer (20 μl) and incubated with the ³²P-MEF-1 probe for 15 min. Thereafter GST-204 (1 μg) was added to the reaction mixture, and it was incubated for another 20 min and analyzed by gel electrophoresis. (C) Transfection of a Flag-Id2 expression plasmid inhibits the binding of C2C12 nuclear proteins to the MEF-1 oligodeoxynucleotide, and induced p204 overcomes the inhibition. EMSA was performed using 10 μg of nuclear extract proteins from the indicated cells cultured in growth medium and the ³²P-MEF-1 oligodeoxynucleotide. L, EMSA with no cell extract (lane 1), with nuclear extract (NE) from C2C12 control cells (lane 2), and with nuclear extract from C2C12 control cells but supplemented with anti MyoD antiserum (lane 3). M, nuclear extract from control cells which had been transfected with Flag vector (lane 4) and nuclear extract from control cells which had been transfected with Flag-Id2 plasmid (lane 5). R, the Muristerone (Mur.)-inducible line (ind.p204/Id2) and the control line (con./Id2) were incubated in growth medium in the absence or presence of 2.5 μM Muristerone for 48 h. (The effects of this incubation on the p204 levels in the two cell lines are shown in panel D.) EMSA was performed using 10 μg of nuclear extract proteins and the ³²P-MEF-1 oligodeoxynucleotide. EMSA with nuclear extracts from con./Id2 cells (lanes 6 and 7) and ind.p204/Id2 cells (lanes 8 and 9) is shown. (D) Induction of p204 expression by Muristerone in the ind.p204/Id2 line but not in the con./Id2 line. Both lines were incubated in GM in the presence (+) or absence (−) of 2.5 μM Muristerone for 48 h. The effects of this incubation on the p204 level were determined by Western blotting. The p204 band is indicated by an arrow. The fold induction of p204 is shown. For further details, see Materials and Methods.